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228 Current Molecular Pharmacology, 2023, 16, 228-233

RESEARCH ARTICLE

Reduction of Genotoxicity of Carbamazepine to Human Lymphocytes by

Pre-treatment with Vitamin B12

Eman K. Hendawi1, Omar F. Khabour2,*, Laith N. AL-Eitan1, and Karem H. Alzoubi3,4

1
Department of Applied Biological Sciences, Faculty of Science, Jordan University of Science and Technology, Irbid
22110, Jordan; 2Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, Jordan University
of Science and Technology, Irbid 22110, Jordan; 3Department of Pharmacy Practice and Pharmacotherapeutics, Uni-
versity of Sharjah, Sharjah, UAE; 4Department of Clinical Pharmacy, Jordan University of Science and Technology,
Irbid, Jordan

Abstract: Background: Carbamazepine (CBZ) is widely used as an anti-epileptic drug. Vitamin

B12 has been shown to protect against DNA damage caused by several mutagenic agents.

ARTICLE HISTORY
Received: November 18, 2021
Revised: February 9, 2022
Accepted: March 2, 2022
DOI:
10.2174/1874467215666220420135924
Keywords: Carbamazepine, vitamin B12, sister chromatid exchanges, DNA damage, chromosomal aberrations, genotoxicity,
white lymphocytes.
Objective: This study aimed to investigate the effect of vitamin B12 on CBZ-induced genotoxicity
in cultured human lymphocytes.
Methods: Sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) genotoxic assays
were utilized to achieve the study objective.
Results: The results showed significantly higher frequencies of CAs and SCEs in the CBZ-treated
cultures (12 μg/mL) compared to the control group (P<0.01). The genotoxic effects of CBZ were
reduced by pre-treatment of cultures with vitamin B12 (13.5µg/ml, P<0.05). Neither CBZ nor vita-
min B-12 showed any effects on mitotic and proliferative indices.
Conclusion: CBZ is genotoxic to lymphocyte cells, and this genotoxicity can be reduced by vitamin
B12.

1. INTRODUCTION Among the side effects of CBZ are ataxia, dizziness,

Epilepsy is regarded as a grave disease marked by peri-
odic and unexpected episodes of seizures attributed to the
imbalance in the excitatory and inhibitory pathways in the
central nervous system [1]. Antiepileptic drugs are common-
ly used drugs to prevent seizures associated with epilepsy
[2]. Among approved antiepileptic drugs by the United
States Food and Drug Administration is carbamazepine
(CBZ) [3, 4]. CBZ can also be used for the treatment of other
neurological conditions, such as bipolar disorder, and the
management of pain associated with some conditions, such
as diabetes and trigeminal neuralgia [3, 5, 6]. CBZ is also a
potent complement receptor 3 (CR3) I-domain-blocking drug
that can interfere with HIV-1 transmission in women [7].
CBZ mediates its therapeutic actions via blocking voltage-
gated sodium channels and the subsequent prevention of the
continuous firing of neurons [8].
*
Address correspondence to this author at the Department of Medical La-
boratory Sciences, Faculty of Applied Medical Sciences, Jordan University
of Science and Technology, Irbid 22110, Jordan; Tel: 0790453765;
Email: khabour@just.edu.jo
drowsiness [4, 9], immune suppression [10], and long-lived
immunological effects in resolved Stevens-Johnson syn-
drome and toxic epidermal necrolysis [11]. In addition, CBZ
has been shown to be genotoxic in different systems, includ-
ing human lymphocytes [12, 13], liver cells [14], human
embryonic stem cells [15], root cells of A. cepa [16], and
others [17].
Vitamin B12, also known as cobalamin, is an essential
nutrient that is naturally present in food and available as a
supplement/prescription medication [18]. Vitamin B12 plays
an important role in many body pathways [19]. For example,
B12 acts as a coenzyme in folate metabolism and for the
biosynthesis of DNA nucleotides [20]. Several studies have
proven the role of vitamin B12 in the integrity of the human
genome [21-23]. Vitamin B12 is required for proper DNA
methylation and reduction of incorporation of uracil into
DNA during replication [21]. Vitamin B12 deficiency is as-
sociated with an elevation of chromosomal damage [24],
induction of micronuclei [25-27], and oxidative stress [28].
In the current study, we investigated the impact of vitamin

1874-4672/23 $65.00+.00 © 2023 Bentham Science Publishers

Reduction of Genotoxicity of Carbamazepine
B12 on the genotoxic effects of CBZ in cultured lymphocyte
cells.
2. METHODS
2.1. Subjects
Healthy adult males were selected to donate blood in the
study. All donors were < 30 years old and did not use sup-
plements, tobacco, or alcohol for at least 6 months prior to
blood sampling [29]. The study was ethically approved by
the Deanship of Research at Jordan University of Science
and Technology (IRB approval number: 29-130-2020 dated
02-02-2020). All procedures were conducted according to
the principles of the Declaration of Helsinki.
2.2. Reagents Used
The culture medium and McIlvaine buffer were pur-
chased from Gibco Invitrogen (UK). Colchicine was pur-
chased from EuroClone (Italy). CBZ and vitamin B12 were
from Sigma-Aldrich (USA).
2.3. Blood Cultures
Cell cultures were established by mixing whole blood (1
mL) with PB-MAX medium (9 mL) in conical culture vials.
For the SCEs assay, cells were treated with 5-
bromdeoxyuridine (20 µg/mL) during culture establishment
[30]. Cultures were incubated at 37°C/5% CO2 for 72 hrs.
The study included 4 groups: Control, CBZ (12 ug/mL), Vit-
amin B12 (13.5 µg/mL), and CBZ + vitamin B12. Cultures
were treated with vitamin B12 directly after the establish-
ment of cultures. Cells were exposed to CBZ for 24 hrs prior
to cell harvesting.
2.4. Cell Harvesting
After incubation of cultures for 72 h, harvesting of meta-
phases was initiated by adding colchicine (10 µg/mL) for 2
hours at 37°C [31]. After treatment with colchicine, cells
were subjected to 0.075 M KCl for 0.5 h, followed by three
washes with the fixative (3X methanol: 1X acetic acid).
Fixed cells were spotted on glass slides and subjected to
staining, as indicated below.
2.5. Analysis of Chromosomal Aberrations
Spotted metaphase spreads were subjected to 5% Giemsa
stain in distilled water. A total of 500 metaphases were
scored per treatment for the presence of chromosomal aber-
rations as previously described. Both breaks and exchanges
were included in the analysis [32].
2.6. Analysis of Sister-Chromatid Exchange
Spotted metaphases were subjected to differential stain-
ing using Hoechst/Giemsa technique. In brief, the slides
were immersed in Hoechst (10 ug/mL) for 30 minutes, ex-
posed to UV light in McIlvaine buffer (ph: 8.3) at 50°C for
20 minutes, and then immersed in 5% Giemsa for 5 minutes.
A total of 250 cells in the second metaphase stage per each
treatment were scored for the presence of sister-chromatid
exchange frequencies [33, 34].
Current Molecular Pharmacology, 2023, Vol. 16, No. 2 229
2.7. Analysis of Mitotic and Proliferative Indices
Mitotic index was computed by calculating the percent-
age of cells in metaphases over the total number of cells
scanned in the microscopic field. At least one thousand cells
per treatment per donor were scored [35]. The proliferative
index was computed by calculating the number of metaphas-
es in the first, second, and third/fourth stages. The formula
for computing the proliferative index was as previously de-
scribed [36].
2.8. Data Processing
Multiple comparisons were performed using ANOVA
followed by Dunnett's post-hoc test. All illustrations were
prepared using GraphPad Prism software. For statistical sig-
nificance, P values <0.05 were considered.
3. RESULTS
The frequencies of chromosomal aberrations induced by
different treatments in cultured lymphocytes are presented in
(Fig. 1). CBZ induced an increase in the frequency of chro-
mosomal aberrations (0.240 ± 0.031 in CBZ versus 0.048 ±
0.015 in control, P<0.01). Treatment of cells with vitamin
B12 did not induce changes in chromosomal aberration lev-
els (0.034 ± 0.009 in vitamin B12 versus 0.048 ± 0.015 in
control, P>0.05). However, vitamin B12 significantly low-
ered chromosomal aberrations induced by CBZ by approxi-
mately 63% (0.240 ± 0.031 in CBZ versus 0.088 ± 0.018 in
CBZ+ vitamin B12, P<0.01, Fig. (1). An example of a
chromosomal break is shown in Fig. (2A).
Fig. (1). The effects of carbamazepine (CBZ) and vitamin B12 on
chromosomal damage in lymphocyte cells. CBZ increased chromo-
somal aberration frequencies. Vitamin B12 did not modulate nor-
mal levels of chromosomal aberrations. However, vitamin B12
lowered chromosomal aberration levels induced by CBZ. *indicates
statistical differences from the control (P<0.01). (A higher resolu-
tion/colour version of this figure is available in the electronic copy
of the article).
A similar result of chromosomal aberrations was ob-
tained with sister-chromatid exchange (Fig. 3). CBZ induced
a significant increase in the frequency of sister-chromatid
exchange (5.08 ± 0.09 in CBZ versus 3.54 ± 0.08 in control,
P<0.01). Treatment of cells with vitamin B12 did not change
sister-chromatid exchange levels (3.45 ± 0.08 in vitamin B12
versus 3.54 ± 0.08 in control, P>0.05). However, vitamin
230 Current Molecular Pharmacology, 2023, Vol. 16, No. 2
B12 significantly lowered sister-chromatid exchanges in-
duced by CBZ by approximately 21% (5.08 ± 0.09 in CBZ
versus 4.04 ± 0.07 in CBZ+ vitamin B12, P<0.01, Fig. 3).
Examples of sister-chromatid exchanges are shown in Fig.
(2B).
Fig. (2). Genotoxicity of CBZ in cultured human lymphocytes. A)
The arrow points to a chromosomal break induced by CBZ. B)
Arrows point to sister chromatid exchanges induced by CBZ. Mag-
nification: 1000X. (A higher resolution/colour version of this figure
is available in the electronic copy of the article).
Fig. (3). The effects of carbamazepine (CBZ) and vitamin B12 on
sister chromatid exchange in lymphocyte cells. CBZ increased sis-
ter-chromatid frequencies. Vitamin B12 did not modulate normal
levels of sister chromatids. However, vitamin B12 lowered sister
chromatids induced by CBZ. * indicates statistical differences from
the control (P<0.01). (A higher resolution/colour version of this
figure is available in the electronic copy of the article).
Hendawi et al.
The effects of the different treatments on the mitotic in-
dex are shown in Fig. (4). None of the treatments affected
the mitotic index (P>0.05). Similar to the mitotic index, none
of the treatments affected the proliferative index Fig. (5),
(P>0.05).
Fig. (4). The impact of carbamazepine (CBZ) and vitamin B12 on
the mitotic index. No significant differences in the mitotic index
between different groups were detected (P>0.05). (A higher resolu-
tion/colour version of this figure is available in the electronic copy
of the article).
Fig. (5). The impact of carbamazepine (CBZ) and vitamin B12 on
the proliferative index. No significant differences in the prolifera-
tive index between different groups were detected (P>0.05). (A
higher resolution/colour version of this figure is available in the
electronic copy of the article).
4. DISCUSSION
In the current study, the modulation of genotoxicity of
CBZ by vitamin B12 was examined using cultured lympho-
cytes. CBZ induced significant increases in chromosomal
aberrations and sister-chromatid exchange. Vitamin B12
significantly lowered the chromosomal damage induced by
CBZ.
The genotoxicity of CBZ is well documented. Exposure
of pregnant women to CBZ can increase the risk of having
children with congenital anomalies, such as neural tube
anomalies, cleft lip and palate, and growth retardations [37-
39]. In addition, CBZ has been reported to induce transcrip-
tion of target genes involved in the regulation of the cell cy-
cle by inhibiting histone deacetylases and subsequent chro-
Reduction of Genotoxicity of Carbamazepine
matin remodeling [40, 41]. An early in vivo study reported a
significant elevation in the frequency of SCE in the cells of
patients medicated with CBZ [42]. In the in vitro cultured
human cells treated with CBZ, a dose-dependent increase in
CA and SCEs treated with CBZ was reported [12]. CBZ was
reported to be genotoxic to Drosophila melanogaster at high
doses [43]. In genotoxicity models of plants, CBZ at envi-
ronmentally relevant concentrations is genotoxic, cytotoxic,
and elevates markers of oxidative stress in root meristem
cells of Allium cepa [16]. The results of the present study
and previous findings using plant, animal, and human mod-
els [15, 44] indicate a strong genotoxic potential of CBZ.
The current results showed a lack of cytotoxicity of CBZ
in cultured human lymphocytes at the examined concentra-
tion. This was demonstrated using the mitotic and the prolif-
erative indices. At therapeutic doses, the toxicity of CBZ was
confined to the nervous and gastrointestinal systems with
side effects that include ataxia, dizziness, drowsiness, cuta-
neous eruptions, hypersensitivity, and others [7, 9, 11, 45].
CBZ has also been shown to induce apoptotic cell death in
cultured cerebellar granule cells [46]. Thus, CBZ might be
cytotoxic to some but not all types of human cells.
The results of this study showed that the genotoxic ef-
fects of CBZ can be ameliorated by vitamin B12. This is
consistent with other studies that showed a protective effect
of vitamin B12 against chromosomal damage induced by
ifosfamide in mice somatic/germ cells [47]. In addition, vit-
amin B12 has been shown to ameliorate chromosomal dam-
age induced by several drugs, such as pioglitazone, paclitax-
el, and thimerosal [29, 48, 49], and disease conditions, such
as kidney failure [50]. Vitamin B12 supplementation has
been shown to bring down the recurrence of micronuclei and
can recuperate the expansion ability of ribavirin-treated cells
[51]. Thus, vitamin B12 can be used to protect human cells
against a spectrum of mutagenic agents.
The mechanisms by which vitamin B12 ameliorates
chromosomal damage associated with mutagenic agents need
to be studied. Among the possible mechanisms is the ability
of vitamin B12 to act as an antioxidant and to normalize ox-
ygen free radicals produced by mutagenic agents [49, 50].
For instance, the free radicals induced by arsenic were re-
ported to diminish after vitamin B12 dietary intake [52].
Other mechanisms might involve the important role that vit-
amin B12 plays in the biosynthesis of DNA nucleotides and
in maintaining the integrity of the human genome [20, 23].
CONCLUSION
In conclusion, Vitamin B12 can ameliorate the genotoxi-
city associated with CBZ in cultured human cells. More in
vivo studies are required to confirm the present findings.
LIST OF ABBREVIATIONS
CAs = Chromosomal aberrations
CBZ = Carbamazepine
IRB = Institutional Review Board
SCEs = Sister-chromatid exchanges
UV = Ultraviolet
Current Molecular Pharmacology, 2023, Vol. 16, No. 2 231
AUTHORS' CONTRIBUTIONS
Experimental design: all authors. Proposal writing and
fund acquisition: E.K.H., K.H.A. and L.N.A. Experimental
work: E.K.H. and O.F.K. Data analysis and interpretations:
all authors. Drafting the manuscript: E.K.H and O.F.K. Edit-
ing and approval of the final draft: all authors.
ETHICS APPROVAL AND CONSENT TO PARTICI-
PATE
The study was ethically approved by the Deanship of Re-
search at the Jordan University of Science and Technology
(IRB approval number: 29-130-2020 dated 02-02-2020).
HUMAN AND ANIMAL RIGHTS
No animals were used in this study. All human proce-
dures were followed in according to the principles of the
Declaration of Helsinki.
CONSENT FOR PUBLICATION
Not applicable.
AVAILABILITY OF DATA AND MATERIALS
The dataset that support the results and funding of this
research are available from the corresponding author.
FUNDING
The authors would like to thank the Jordan University of
Science and Technology for providing support to conduct the
study (grant: 123/2020).
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
Declared none.
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